Isolation of single nuclei from solid tissues

Blue Lake; Kun Zhang

Oct 24, 2019

Isolation of single nuclei from solid tissues

DOI

dx.doi.org/10.17504/protocols.io.ufketkw

Blue Lake¹,
Kun Zhang¹

¹University of California, San Diego

Human Cell Atlas Method Development Community
KPMP

Blue Lake

UCSD

DOI: dx.doi.org/10.17504/protocols.io.ufketkw

External link: http://genome-tech.ucsd.edu/ZhangLab/

Protocol Citation: Blue Lake, Kun Zhang 2019. Isolation of single nuclei from solid tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.ufketkw

Manuscript citation:

Lake, B.B. et al. A single-nucleus RNA-sequencing pipeline to decipher the molecular anatomy and pathophysiology of human kidneys. Nature Communications 10, 2832 (2019).

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: October 08, 2018

Last Modified: October 24, 2019

Protocol Integer ID: 16588

Keywords: single nuclei, frozen tissues, genomic assays

Abstract

Nuclei can be readily isolated from frozen tissues with a combination of chemical and physical treatments that can circumvent the non-uniform or incomplete dissociation of solid tissues into single cells. The isolation of nuclei can also circumvent RNA degradation or any introduction of technical artefacts (such as stress responses) that could be triggered during whole cell dissociation methods. Data generated from single-nucleus genomic assays permits discovery of molecular cell types that can be used to define the overall cellular makeup of a tissue or organ, and ultimately will inform upon adult human tissue atlases.

Materials

MATERIALS
ReagentDAPI Thermo Fisher ScientificCatalog #D1306 
ReagentDounce homogenizers Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8938-1SET 
ReagentRNAse InhibitorEnzymaticsCatalog #Y9240L 
ReagentCellTrics Filters (30um)SysmexCatalog #04-004-2326 
STEP MATERIALS
ReagentRNase ZapMerck MilliporeSigma (Sigma-Aldrich)Catalog #R2020-250ML 
ReagentRNAlaterThermo Fisher ScientificCatalog #AM7020 
ReagentRNAse InhibitorEnzymaticsCatalog #Y9240L 
ReagentcOmplete™, Mini Protease Inhibitor CocktailRocheCatalog #11836153001 
ReagentDAPIInvitrogen - Thermo FisherCatalog #D3571 
ReagentRNAlaterThermo Fisher ScientificCatalog #AM7020 

Protocol materials

ReagentRNAse InhibitorEnzymaticsCatalog #Y9240L 
ReagentCellTrics Filters (30um)SysmexCatalog #04-004-2326 
ReagentRNAlaterThermo Fisher ScientificCatalog #AM7020 
ReagentRNAse InhibitorEnzymaticsCatalog #Y9240L 
ReagentcOmplete™, Mini Protease Inhibitor CocktailRocheCatalog #11836153001 
ReagentDAPIInvitrogen - Thermo FisherCatalog #D3571 
ReagentDAPI Thermo Fisher ScientificCatalog #D1306 
ReagentDounce homogenizers Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8938-1SET 
ReagentRNase ZapMerck MilliporeSigma (Sigma-Aldrich)Catalog #R2020-250ML 
ReagentRNAlaterThermo Fisher ScientificCatalog #AM7020 
ReagentDAPIInvitrogen - Thermo FisherCatalog #D3571 
ReagentRNAse InhibitorEnzymaticsCatalog #Y9240L 
ReagentcOmplete™, Mini Protease Inhibitor CocktailRocheCatalog #11836153001 
ReagentRNase ZapMerck MilliporeSigma (Sigma-Aldrich)Catalog #R2020-250ML 
ReagentRNAlaterThermo Fisher ScientificCatalog #AM7020 
ReagentRNAlaterThermo Fisher ScientificCatalog #AM7020 

Prepare Reagents and Tissue

Prepare NEB-complete (NEB containing 5 ug/ml DAPI and 0.04 U/ul RNAse Inhibitor)

chill on ice


Final ConcentrationStockVolume (25 ml)
20 mM Tris [pH 8]
  320 mM sucrose 
  5 mM CaCl2 
  3 mM MgAc2
  0.1 mM EDTA
  0.1% TritonX-100
  dH201M 
  1M 
  1M 
  1M 
  0.5M 
  10%
  -0.5ml
  8ml
  125µl
  75µl
  5µl
  250µl
  16ml
NEB Base Solution Composition


Note
For chromatin accessibility assays, include 1:100 dilution of cOmplete™ Protease Inhibitor Cocktail (stock one tablet in 0.5 ml H2O) 


ReagentDAPIInvitrogen - Thermo FisherCatalog #D3571 

ReagentRNAse InhibitorEnzymaticsCatalog #Y9240L 

ReagentcOmplete™, Mini Protease Inhibitor CocktailRocheCatalog #11836153001 

Treat dounce with RNAseZap, rinse with sterile water (if possible: UV treat Duration00:15:00  )

ReagentRNase ZapMerck MilliporeSigma (Sigma-Aldrich)Catalog #R2020-250ML

Transfer vial containing tissue to ice.

Note
For solid tissues (e.g. adult human kidney), 40 µm cryosections can be used with the number of sections dependendent on desired yield and the size and type of tissue. For kidney ~6 cubic mm will give ~150-200K nuclei.

For sections stored in a stabilizing solution (e.g. RNAlater), wash briefly with PBS and immediately proceed to Step 5 below


ReagentRNAlaterThermo Fisher ScientificCatalog #AM7020 


Final ConcStockVolume (50ml)
1x PBS 
  1 mM EGTA
  dH2O10x
  0.1M5ml
  50µl
  45ml
PBSE Composition

Isolate Nuclei

Add Amount1 mL   ice cold NEB buffer to tissue segments

Cut end off of a p1000 tip to increase bore size using a sterile scalpel, then pipette sections up and down to disperse and dissolve OCT, ~20 times 

Using a regular p1000 tip, pipette ~10x to further dissociate tissues into manageable sizes. 

Note
tissue needs to be passable through a p1000 tip easily before proceeding

Then transfer to dounce homogenizer

Gently dounce tissue on ice: 

5 strokes with pestle A

~20 strokes with pestle B (minimize bubble formation) 


Note
Increase number of pestle A strokes if the tissue appears too granular before proceeding with pestle B. Number of pestle B strokes used here is dependent on tissue toughness:
soft tissues use ~10-15 strokes
hard tissues use ~15-20 strokes


Note
Avoid making bubbles

Transfer solution to a Amount15 mL   tube

Wash dounce with Amount1 mL  NEB-complete buffer and add this into the same tube

Incubate on ice Duration00:10:00  

Pass supernatant through 30 uM CellTrics filter to a new Amount15 mL   conical tube


Equipment
new equipment
NAME
Sysmex
BRAND
04-004-2324
SKU
30 uM Celltrics Filter
SPECIFICATIONS

Bring up to Amount10 mL   with PBSE

Pellet nuclei: Amount900 g   
Duration00:10:00   at Temperature4 °C  

snRNA-Seq methods: nuclei can be stored in RNAlater

Remove supernatant and resuspend pellet in Amount100 µL   - Amount1000 µL    PBS + 0.1% RNAse Inhibitor

Note
Resuspension buffer and volume is dependent on downstream assays and nuclei concentration requirements. 1% BSA can be included here

QA/QC: Count nuclei (e.g. BioRad T20 Cell Counter)


Equipment
new equipment
NAME
Bio-Rad
BRAND
1450011
SKU
Cell Counting Slides for TC10™/TC20™ Cell Counter, Dual-Chamber
SPECIFICATIONS

 QA/QC: Check nuclei integrity under fluorescent microscope using DAPI channel. Nuclei should appear distinct, have rounded borders and the majority occurring as singlets. 

Note
High clumping rates would indicate damaged nuclei and would require re-filtering using 30-µm CellTrics filter or exclusion from downstream analyses. 

At least 50,000 nuclei are needed to proceed with snDrop-seq

At least 10,000 nuclei are needed to proceed with 10X 3’ RNA v3

Isolate Nuclei

To use nuclei directly for single nucleus assays, proceed to method 

To use nuclei on a later date, proceed to Step 19  

snRNA-Seq methods: nuclei can be stored in RNAlater

Add Amount900 µL   RNAlater to Amount100 µL   nuclei in PBS, incubate at Temperature4 °C   for Duration01:00:00   to Duration02:00:00  

then transfer to Temperature-20 °C   for 1-2 months
ReagentRNAlaterThermo Fisher ScientificCatalog #AM7020 

To remove RNAlater, centrifuge nuclei at 4000g, Duration00:10:00   at Temperature4 °C  . 

Remove solution and resuspend in associated nuclei resuspension buffer (assay dependent)

	Final Concentration	Stock	Volume (25 ml)
	20 mM Tris [pH 8] 320 mM sucrose 5 mM CaCl2 3 mM MgAc2 0.1 mM EDTA 0.1% TritonX-100 dH20	1M 1M 1M 1M 0.5M 10% -	0.5ml 8ml 125µl 75µl 5µl 250µl 16ml

	Final Conc	Stock	Volume (50ml)
	1x PBS 1 mM EGTA dH2O	10x 0.1M	5ml 50µl 45ml

Public workspaceIsolation of single nuclei from solid tissues

Isolation of single nuclei from solid tissues